Compositions and methods for in situ single cell analysis using enzymatic nucleic acid extension

Abstract

The present disclosure is based in part on probes, compositions, methods, and kits for simultaneous, multiplexed spatial detection and quantification of protein and/or nucleic acid expression in a user-defined region of a tissue, user-defined cell, and/or user-defined subcellular structure within a cell.

What is claimed is: 
     
         1 . A method for in situ synthesis of a nucleic acid sequence in a tissue sample, the method comprising:
 a) contacting the tissue sample with at least one probe,
 wherein the probe comprises a target-binding domain and a target identification domain, 
 wherein the probe comprises a free 3′-OH moiety, and 
 wherein the target-binding domain binds to at least one target molecule located at a first location of the tissue sample; 
   b) contacting the tissue sample with at least one reversible terminator nucleotide, at least one polymerase, at least one caged chelator-cofactor complex, and at least one unbound caged chelator,
 wherein the at least one caged chelator-cofactor complex comprises at least one cofactor bound to a caged chelator; 
 wherein the at least one reversible terminator nucleotide comprises the nucleotide operably linked to a cleavable 3′ terminator moiety; 
   c) illuminating the first location of the tissue sample with light sufficient to uncage the at least one caged chelator-cofactor complex, thereby releasing the at least one cofactor,   thereby activating the at least one polymerase,   thereby ligating the at least one reversible terminator nucleotide to the free 3′-OH moiety of the at least one bound probe;   d) washing the tissue sample;   e) treating the tissue sample under conditions sufficient to cleave the 3′ terminator moiety of the at least one reversible terminator nucleotide ligated in step (c), thereby exposing a free 3′-OH moiety on the extended at least one bound probe; and   f) repeating steps (b)-(e) until the nucleic acid sequence has been synthesized.   
     
     
         2 . The method of  claim 1 , wherein the target-binding domain binds to at least one target molecule at an at least second location of the tissue sample, and wherein the method further comprises repeating steps (b)-(f) at the at least second location. 
     
     
         3 . The method of  any one of the preceding claims , wherein the nucleic acid sequence synthesized at the first location of the tissue sample is different than the nucleic acid sequence synthesized at the at least second location of the tissue sample. 
     
     
         4 . A method of producing a spatially-resolved profile of the abundance of at least two target analytes in a first and an at least second location of a tissue sample comprising:
 a) contacting the tissue sample with a solution comprising at least two species of probes, the probes comprising a target-binding domain and a target identification domain,   wherein each species of probe comprises a unique target-binding domain that binds to one of the at least two target analytes and a unique target identification domain specific for the target analyte, and a free 3′-OH moiety;   b) contacting the tissue sample with a first plurality of reversible terminator nucleotides, a first plurality of polymerases, a first plurality of caged chelator-cofactor complexes, and a first plurality of unbound caged chelator,
 wherein at least one caged chelator-cofactor complex in the first plurality comprises at least one cofactor bound to a caged chelator; 
 wherein at least one reversible terminator nucleotide in the first plurality comprises the nucleotide operably linked to a cleavable 3′ terminator moiety; 
   c) illuminating at least one location of the tissue sample with light sufficient to uncage the caged chelator-cofactor complexes, thereby releasing the cofactors at the at least one location of the tissue sample,   thereby activating the polymerases at the at least one location of the tissue sample   thereby ligating a reversible terminator nucleotide to the free 3′-OH moiety of at least one probe bound to a target analyte at the at least one location of the tissue sample, thereby extending the at least one bound probe;   d) washing the tissue sample;   e) treating the tissue sample under conditions sufficient to cleave the 3′ terminator moiety of the at least one reversible terminator nucleotide ligated in step (c), thereby exposing a free 3′-OH moiety on the extended at least one bound probe;   f) repeating steps (b)-(e) until   at least one probe bound to a target analyte in the first location of the tissue sample has been extended such that a first spatial barcode domain has been synthesized, wherein the first spatial barcode domain comprises a unique nucleic acid sequence specific to the first location of the tissue sample,   at least one probe bound to a target analyte in the at least second location of the tissue sample has been extended such that a second spatial barcode domain has been synthesized, wherein the second spatial barcode domain comprises a unique nucleic acid sequence specific to the at least second location of the tissue sample;   g) collecting the probes bound to target analytes in the tissue sample; and   h) quantifying via sequencing the probes collected in step (f), thereby determining the abundance of the at least two target analytes in the first and the at least second location of the tissue sample, thereby producing a spatially-resolved profile of the abundance of the at least two target analytes.   
     
     
         5 . The method of claim  5  or claim  6 , further comprising comparing the abundance of the at least two target analytes in the first location of the tissue sample and the at least two target analytes in the at least second location of the tissue sample. 
     
     
         6 . The method of  any one of the preceding claims , wherein the polymerase is terminal deoxynucleotidyl transferase or a biologically active fragment thereof. 
     
     
         7 . The method of  any one of the preceding claims , wherein the cleavable 3′ terminator moiety is a 3′-ONH 2  group. 
     
     
         8 . The method of  any one of the preceding claims , wherein the caged-chelator is a caged divalent cation chelator, preferably wherein the caged-chelator is 1-(4,5-Dimethoxy-2-Nitrophenyl)-1,2-Diaminoethane-N,N,N′,N′-Tetraacetic Acid (DMNP-EDTA). 
     
     
         9 . The method of  any one of the preceding claims , wherein the cofactor is a divalent metal cofactor, preferably wherein the cofactor is Co 2+ , Mg 2+ , Mn 2+  Ca 2+ , Cd 2+ , Zn 2+  or Fe 2+ , preferably wherein the cofactor is Co 2+ . 
     
     
         10 . The method of  any one of the preceding claims , wherein the light sufficient to uncage the caged chelator-cofactor complex is UV light. 
     
     
         11 . The method of  any one of the preceding claims , wherein treating the tissue sample under conditions sufficient to cleave the 3′ terminator moiety of the at least one reversible terminator nucleotide comprises treating the tissue sample under acidic conditions, preferably wherein treating the tissue sample under acidic conditions comprises contacting the tissue sample with a solution with a pH of about 5.5. 
     
     
         12 . The method of  any one of the preceding claims , wherein the probes further comprise:
 i) a unique molecular identifier;   ii) an amplification primer binding site;   iii) a constant region; or   iv) any combination of the preceding,   preferably wherein the probes comprise, from 5′ to 3′, the target binding domain, followed by the amplification primer binding site, followed by the unique molecular identifier, followed by the target identification domain, followed by the constant region.   
     
     
         13 . The method of  any one of the preceding claims , wherein the spatial barcode domain of at least one probe bound to a target analyte in the first location of the tissue sample comprises a unique spatial identifier sequence specific to the first location of the tissue sample, and/or
 the spatial barcode domain of at least one probe bound to a target analyte in the at least second location of the tissue sample comprises a unique spatial identifier sequence specific to the at least second location of the tissue sample.   
     
     
         14 . The method of  any one of the preceding claims , wherein the spatial identifier sequence comprises
 i) at least about 20 nucleotides; or   ii) at least about 28 nucleotides.   
     
     
         15 . The method of  any one of the preceding claims , wherein the spatial identifier sequence comprises at least four spatial identification domains, preferably wherein
 i) each of the at least four spatial identification domains comprise the same number of nucleotides; or   ii) at least one of the at least four spatial identifications domains comprise a different number of nucleotides as compared to another spatial identification domain within the same spatial barcode.   
     
     
         16 . The method of  any one of the preceding claims , wherein each spatial identification domain comprises about 1 to about 4 nucleotides, preferably wherein each spatial identification domain comprises about 4 nucleotides. 
     
     
         17 . The method of  any one of the preceding claims , wherein each of the at least four spatial identification domains comprise the same nucleotide at the 3′ terminus 
     
     
         18 . The method of  any one of the preceding claims , wherein the method further comprises, after step (f) and prior to step (g), repeating steps (b)-(e) to extend the spatial barcode domain in each location of the tissue sample such that the spatial barcode domain comprises, at the 3′ end, a delimiting domain. 
     
     
         19 . The method of  any one of the preceding claims , wherein the method further comprises, after step (f) and prior to step (g), extending the spatial barcode domain in each location of the tissue sample such that the spatial barcode domain comprises a polyT domain. 
     
     
         20 . The method of  any one of the preceding claims , wherein the method further comprises, after step (f) and prior to step (g):
 (i) repeating steps (b)-(e) to extend the spatial barcode domain in each location of the tissue sample such that the spatial barcode domain comprises, a delimiting domain; and   (ii) extending the spatial barcode domain in each location of the tissue sample such that the spatial barcode domain comprises a polyT domain.   
     
     
         21 . The method of  any one of the preceding claims , wherein the sequence of the delimiting domain is the same for every spatial barcode in the sample. 
     
     
         22 . The method of  any one of the preceding claims , wherein the first location of the tissue sample and the at least second location of the tissue sample are subcellular. 
     
     
         23 . The method of  any one of the preceding claims , wherein the first location of the tissue sample and the at least second location of the tissue sample each
 i) comprise no more than one cell; or   ii) comprise no more than ten cells.   
     
     
         24 . The method of  any one of the preceding claims , the method further comprising prior to step (a), subjecting the tissue sample to ddTTP (dideoxthymidine-triphosphate) termination, preferably wherein subjecting the tissue sample to ddTTP termination comprises contacting the tissue sample with ddTTP and TdT. 
     
     
         25 . The method of  any one of the preceding claims , the method further comprising after step (g) and prior to step (h), amplifying the collected probes, preferably wherein amplifying the collected probes comprises the use of a first amplification primer and a second amplification primer, wherein
 the first amplification primer comprises a first flow cell adapter sequence, a first NGS index sequence and a first sequencing primer binding site, and   the second amplification primer comprises a second flow cell adapter sequence, a second NGS index sequence and second sequencing primer binding site.   
     
     
         26 . The method of  any one of the preceding claims , wherein at least one of the first and the second amplification primers comprises a nucleic acid sequence that is complementary to the delimiting sequence and/or the polyT domain.